It was not long ago that the war against microbial disease was thought to be won, through the use of antibiotics and vaccines. It is now seen that there remains much to be done in this field. Although antibiotics are a huge industry, totaling $22.9 billion in worldwide sales, there are still significant unmet clinical needs caused by growing bacterial resistance problems that require new antibacterial therapies.
A bacterial pathogen of major concern is Mycobacterium tuberculae (M. tb.). It is highly infectious, and treatment requires a long course of antibiotics. Frequently, patients fail to complete treatment, thereby selecting for antibiotic resistant bacteria. Significant reservoirs of virtually untreatable M.tb. are already present in some lesser developed parts of the world. It is expected that these will cause a pandemic in the future, unless improved therapies can be produced.
The mammalian immune system plays a key role in controlling the course of microbial infection in vivo. T cells play a pivotal role as regulators and effectors in the immune response. Cytolytic T cells (CTL) are directly involved in the lysis of foreign and virally infected cells. Two major types of T cell-mediated cytolysis have been well characterized. The regulated, directional release of cytoplasmic granule contents is a primary mechanism by which CTL and NK cells initiate the target cell death process. In addition, Fas-Fas ligand interaction mediates T-cell directed cytolysis.
Proteins present in cytoplasmic granules, including the pore-forming protein perforin, and a family of serine proteases called granzymes, have been implicated in granule mediated cytolysis. Interest in the precise mechanism of cytolysis has stimulated the search for additional molecules present on these highly specialized organelles. Early studies of cytolytic granules included density gradient purification and subsequent analysis, in which were observed low molecular weight proteins of unknown identity. It has recently been determined that one of the proteins present in granules is the late T cell activation marker, granulysin.
Many intracellular and extracellular pathogens meet their death within phagocytes. T cells contribute to antimicrobial defense by activating macrophages to kill the foreign invader. However, a microbicidal pathway by which T cells directly kill the pathogen has not been elucidated. Exploring the pathways by which microbes are eliminated in vivo is of great interest for the development of novel therapies and screening for novel antibiotics
Relevant Literature
U.S. Pat. No. 4,994,369 discloses the nucleotide and predicted amino acid sequence of the xe2x80x9c519xe2x80x9d protein, herein referred to as granulysin. The sequences of granulysin variants may be accessed from the Genbank and EMBL databases, with the accession number X05044 for the mRNA sequence of 519; EMBL accession X05044 for the encoded protein; and EMBL: locus HSNKG5, accession X54101 for the NKG5 splice variant.
The sequence and structure of the granulysin gene is discussed in Jongstra et al. (1987) J. Exp. Med. 165:601-614; Donlon et al. (1990) Cytogenet. Cell Genet. 53:230-231; and Manning et al. (1992) J. Immunol. 148:4036-4042.
The processing, sub-cellular localization and function of granulysin in cell-mediated cytotoxicity is discussed in Pena and Krensky (1997) Sem. Immunol. 9:117-125; and in Pena et al. (1997) J. Immunol. 158:2680-2688.
The structure, function and expression of human and murine perforin is discussed in Podack et al. (1988) Immun. Rev. 103:203-211. The role of perforin in cytolytic T cell killing is explored through the use of transgenic knock-outs in Lowin et al. (1994) P.N.A.S. 91:11571-11575.
Methods are provided for the use of granulysin protein as an antimicrobial agent. A pharmaceutical composition comprising granulysin as an active agent is administered to a patient suffering from a microbial infection, particularly bacterial infections. The protein is also effective at killing a variety of microbial organisms in vitro. Granulysin may be administered alone, or in combination with other bacteriocidal agents, e.g. perforin, antibiotics, etc. Such combined formulations are effective in killing M. tb. The administered granulysin may be one of the naturally occurring forms of the protein, or a synthetic variant derived therefrom. Granulysin mediated killing of microbes is also useful for modeling and screening novel antibiotics.